Use Yeast Two Hybrid experiment to identify Pxa2p COOH-terminus interact with the NBF of Pxa1p and Pxa2p in Saccharomyces cerevisiae

• Main Authors: Shih-Ming, 陳詩銘
• Other Authors: 陳凌雲
• Format: Others
• Language: zh-TW
• Published: 2010
• Online Access: http://ndltd.ncl.edu.tw/handle/71624527890107314281

碩士 === 中山醫學大學 === 生化暨生物科技研究所 === 98 === The Yeast peroxisomal half ABC transporters, Pxa1p and Pxa2p, are the homologous proteins of human ALDP and PMP70. The function of ALDP and PMP70 are understood limitedly. ALDP is thought to be involved in the uptake of activated VLCFAs into mammalian peroxisomes. PMP70 is speculated to be involved in the transportation of LCFA into mammalian peroxisomes. To our knowledge, the half ABC transporters have to dimerize to be functional. In 1999, Liu and co-workers studied the COOH terminus of the human ALDP, ALDRP and PMP70 by Yeast Two Hybrid and co-immunopricipitation experiments to show that the COOH terminus of the human ALDP, ALDRP and PMP70 will form homodimer or heterodimer. Pxa1p and Pxa2p, the peroxisomal membrane proteins, are known to form heterodimer to transport LCFA into peroxisomes. There are no reports about the binding domains between Pxa1p and Pxa2p. Using the Yeast Two hybrid system, we show that the interactions occur between NBF_CT(Pxa2p) and NBF(Pxa2p)or NBF(Pxa2p) but not NBF_CT(Pxa1p). Moreover, the interaction area of the Pxa2p COOH terminus is from 724th to 774th amino acids by the deletion mutants. Finally, using site-directed mutagenesis and beta-galactosidase activity assay, we determine several conserved amino acids could affect the protein-protein interaction.